Rna labeling method

Abstract

A method of sample analysis is provided. In certain embodiments, the method involves: a) obtaining a fragmented RNA sample comprising fragments of long RNA molecules and short RNA molecules; b) ligating an adaptor to an end of the RNA of the fragmented RNA sample to produce an adaptor-ligated sample; c) hybridizing said adaptor-ligated sample to an array of nucleic acid probes; and d) reading said array to obtain an estimate of the abundance of a long RNA in the RNA sample and an estimate of the abundance a small RNA in the RNA sample.

1 . A method of sample analysis comprising:
 a) obtaining a fragmented RNA sample comprising:
 i. fragments of long RNA molecules, wherein the long RNA molecules are at least 200 nucleotides in length; and 
 ii. short RNA molecules of less then 200 nucleotides in length; 
   b) ligating an adaptor to an end of the RNA of said fragmented RNA sample to produce an adaptor-ligated sample;   c) hybridizing said adaptor-ligated sample to an array of nucleic acid probes; and   d) reading said array to obtain an estimate of the abundance of a fragmented long RNA in said RNA sample and an estimate of the abundance of a small RNA in said RNA sample.   
     
     
         2 . The method of  claim 1 , wherein said fragmented RNA sample is made by exposing an initial RNA sample comprising intact long RNA and short RNA to fragmentation conditions. 
     
     
         3 . The method of  claim 2 , wherein said exposing comprises contacting said initial RNA sample with a divalent cation at a temperature of at least 50° C. 
     
     
         4 . The method of  claim 2 , wherein said initial RNA sample comprises total cellular RNA. 
     
     
         5 . The method of  claim 2 , wherein said initial RNA sample comprises total cellular RNA from which tRNA and rRNA has been removed. 
     
     
         6 . The method of  claim 1 , wherein said fragmented RNA sample is obtained from formalin-fixed paraffin embedded tissue (FPET) sample. 
     
     
         7 . The method of  claim 1 , wherein said short RNA molecules comprise small RNA molecules selected from the group consisting of short interfering RNA (siRNA) molecules, microRNA (miRNA) molecules, tiny non-coding RNA (tncRNA) molecules or small modulatory RNA (smRNA) molecules. 
     
     
         8 . The method of  claim 1 , wherein said long RNA molecules comprise lincRNA molecules or mRNA molecules. 
     
     
         9 . The method of  claim 1 , wherein said method comprises ligating an adaptor to the 3′ end of the RNA of said fragmented RNA sample. 
     
     
         10 . The method of  claim 1 , wherein said method comprises ligating an adaptor to the 5′ end of the RNA of said fragmented RNA sample. 
     
     
         11 . The method of  claim 1 , wherein the RNA of said fragmented RNA sample is labeled prior to or after said ligating step b). 
     
     
         12 . The method of  claim 1 , wherein said adaptor is a labeled adaptor. 
     
     
         13 . The method of  claim 1 , wherein said adaptor is in the range of 6 to 12 nucleotides in length. 
     
     
         14 . The method of  claim 1 , further comprising treating said fragmented RNA sample with a polynucleotide kinase or a phosphatase in the absence of ATP prior to said ligating step b). 
     
     
         15 . The method of  claim 1 , wherein the adaptor-ligated sample comprises
 i. adaptor-ligated short RNA comprising an adaptor portion and a short RNA portion; and   ii. adaptor-ligated long RNA fragments comprising an adaptor portion and a long RNA portion;   and wherein said array comprises
 i. a first nucleic acid probe that comprises a nucleotide sequence that is complementary to both said adaptor portion and said short RNA portion of said adaptor-ligated short RNA; and 
 ii. a second probe that comprises a nucleotide sequence that is complementary to the long RNA portion of said adaptor-ligated long RNA but not said adaptor portion of said long RNA. 
   
     
     
         16 . An array comprising:
 i. a plurality of first probes that comprise nucleotide sequences that are complementary to different adaptor-ligated short RNAs, wherein said first probes are complementary to both the adaptor portions and the short RNA portions of said adaptor-ligated short RNAs; and   ii. a plurality of second probes that comprise nucleotide sequences that are complementary to different long RNAs.   
     
     
         17 . The array of  claim 16 , wherein said adaptor is in the range of 6 to 12 nucleotides in length. 
     
     
         18 . The array of  claim 16 , wherein said array comprises at least 100 of said first probes and at least 1000 of the second probes. 
     
     
         19 . A kit comprising:
 a) an array of  claim 16 ;   b) an RNA ligase; and   c) an adaptor.   
     
     
         20 . The kit of  claim 19 , further comprising instructions for performing the method of  claim 1 .